Multicompartmental analysis of the effects of dietary fat saturation and cholesterol on absorptive lipoprotein metabolism in the rat.

The potential contribution of the metabolism of absorptive lipoproteins to effects of dietary cholesterol and saturated (versus polyunsaturated) fat on plasma lipids and lipoproteins was studied in rats. Recipients were fed either a commercial stock diet, a low fat/cholesterol-free semipurified diet with a neutral P/S ratio, or one of two high fat/cholesterol-containing diets that had a high (4.6) or low (0.2) P/S ratio. Chylomicrons and intestinal very low-density lipoproteins labeled with 3H-oleic acid and/or 14C-cholesterol were isolated from donor rats receiving the high or low P/S ratio oil and were injected into recipients. Data on plasma disappearance and hepatic recovery of labels were analyzed by compartmental analysis. A multicompartmental model was required to fit these data and included steps interpreted to correspond to activation of newly secreted intestinal lipoproteins; delipidation on capillary endothelia by lipoprotein lipase, with transfer of cholesterol to other lipoproteins and tissue uptake of cholesterol and fatty acids; hepatic clearance of remnants; and secretion of lipoproteins by the liver. Metabolic state of the recipients (especially plasma triglyceride concentrations) had a greater influence on the rate of chylomicron turnover than did the source of donor chylomicrons, although saturated chylomicrons tended to be metabolized more slowly than polyunsaturated ones. For recipients fed the commercial stock diet and injected with saturated (versus polyunsaturated) chylomicrons, the mechanistic model predicted an increased retention of triglycerides during remnant formation, and thus an increased delivery of triglyceride to the liver. A consequent elevation in hepatic production of very low-density lipoprotein triglycerides may be related to the observed slower clearance of chylomicrons and increased plasma lipid levels in rats fed saturated fat and cholesterol.

Controlling factors in the maintenance of plasma cholesterol concentration in the rabbit.

We have investigated factors important in the maintenance of plasma cholesterol concentration in three groups of New Zealand white rabbits fed either a cholesterol-free, commercial stock diet or an atherogenic diet consisting of stock diet supplemented with 0.1% cholesterol and 10% corn oil. When fed stock diet, the plasma cholesterol concentration in female rabbits (164 mg/100 ml) of one strain was significantly greater than plasma cholesterol concentration in males (67 mg/100 ml) of the same strain or in females (62 mg/100 ml) of another strain. Cholesterol absorption and turnover, steroid excretion and tissue cholesterol storage were examined. The percentage of an oral dose of isotopically labeled cholesterol absorbed could not account for group differences in plasma cholesterol concentrations and was not changed by cholesterol supplementation for 3 weeks. Turnover of plasma cholesterol conformed to a two-pool model in rabbits fed stock diet and the atherogenic diet. In rabbits fed the atherogenic diet, the irreversible disposal rate from pool A and total acidic steroid excretion were inversely related to plasma cholesterol concentration. One controlling factor in the maintenance of relatively low plasma cholesterol concentrations in cholesterol-fed rabbits appears to be enhanced bile acid excretion.

Quantitation of endogenously occupied and unoccupied binding sites for 1,25-dihydroxyvitamin D3 in rat intestine.

The quantitative reversible dissociation of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-receptor complex by the mercurial reagent mersalyl was used to develop an assay for endogenously occupied and unoccupied 1,25-(OH)2D3 binding sites. Incubation of intestinal cytosol preparations in buffer containing 50 mM Tris . HCl, 300 mM KCl, and 1.5 mM EDTA, pH 7.4, with 1 mM mersalyl for 60 min was effective in inhibiting 98% of 1,25-(OH)2D3 specific binding activity. Dissociation of bound 1,25-(OH)2[26,27-3H]D3 from the hormone-receptor complex approached completion by 180 min. In cytosol incubated with saturating levels of nonradioactive hormone, 96% of total binding activity was measurable with the hormone binding assay after displacement of bound nonradioactive ligand with 1 mM mersalyl. Endogenously occupied 1,25-(OH)2D3 binding sites contributed 0, 9, and 19% of total binding activity in rats with plasma 1,25-(OH)2D3 levels averaging 2, 121 +/- 36 and 516 +/- 92 pg/ml, respectively. Therefore, the major fraction of cytosolic 1,25-(OH)2D3 specific binding activity is unoccupied in rat intestine. The results suggest that only a small proportion of the measurable receptors are in the bound form to provide maximal 1,25-(OH)2D3-induced calcium transport.

Glucocorticoids and appearance of 1,25-dihydroxyvitamin D3 receptor in rat intestine.

The ontogenesis of the 1,25-dihydroxyvitamin D3 specific binding activity in intestine was examined in vitamin D-deficient and replete rats. The absence of binding activity in intestines during the first two postnatal weeks was not influenced by vitamin D supplementation. The concentration of binding sites peaked on day 18 in vitamin D-replete rats and preceded that in the deficient group by approximately 1 wk. The influence of glucocorticoids on 1,25-dihydroxyvitamin D3-binding protein levels was examined by sequential hydrocortisone administration and adrenalectomy. Subcutaneous hydrocortisone administration before day 14 postpartum did not induce binding activity. The concentration of binding sites was significantly increased to 369 +/- 60 fmol/mg of protein by hydrocortisone injections from days 15 to 17 postpartum when compared with an average of 182 +/- 16 fmol/mg of protein in littermate controls. Hydrocortisone administration did not further increase receptor levels in rats injected from days 19 to 21. Bilateral adrenalectomy on day 17 postpartum significantly decreased the concentration of binding sites. It is concluded that adrenal glucocorticoids play an important role in the developmental appearance of 1,25-dihydroxyvitamin D3 specific binding activity in the postnatal rat intestine.

Molecular events involved in 1,25-dihydroxyvitamin D3 stimulation of intestinal calcium transport.

There is a biphasic response of intestinal calcium transport to 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). The first or rapid response is by existng mature villus cells, whereas the slow second response is by maturing crypt cells. For both responses, [3H]1,25-(OH)2-D3 localizes in the nucleus before initiating the transport events. This localization is brought about by a specific cytoplasmic receptor, which has a molecular weight of 67,000, is highly specific for 1,25-(OH)2-D3, and has a Kd of 5 X 10(-11) M. Its essentiality for intestinal calcium transport response to 1,25-(OH)2-D3 can be demonstrated in neonatal rat pups. In cultured chick intestinal duodena calcium transport begins to appear within 4 h after the addition of 1,25-(OH)2-D3. The response of this calcium transport system to 1,25-(OH)2-D3 is totally blocked by cycloheximide in a reversible manner. Similarly, it is blocked by actinomycin D in a partially reversible manner. These results make it obvious that the rapid calcium transport response to 1,25-(OH)2-D3 involves nuclear activity and transcription of DNA into functional proteins. The exact nature of the transport proteins remains largely unknown except for the calcium-binding protein originally discovered by Wasserman and colleagues. The transport proteins are believed to operate at the brush border membrane surface to facilitate the transfer of calcium and phosphorus into the absorption cells.

Effects of recombinant interferon-alpha 2 treatment upon lipid concentrations and lipoprotein composition.

Interferon-alpha 2 (IFN-alpha 2) produced by recombinant DNA technology and purified to homogeneity, was assessed for effects on plasma lipids and lipoprotein composition in 10 patients with metastatic malignant melanoma. Patients received 30 X 10(6) U/m2 IFN intravenously for 5 consecutive days every 3 weeks. Plasma cholesterol concentrations were significantly decreased after 3 days of IFN administration (171 +/- 37 mg/dl, mean +/- SD) when compared with pretreatment concentrations (211 +/- 28 mg/dl, p less than 0.05). Approximately 34% of the decrease in plasma cholesterol concentration was contributed by high-density lipoprotein (HDL). Significant decreases in plasma HDL cholesterol (44 +/- 8 to 34 +/- 7 mg/dl, p less than 0.05) and apolipoprotein A-1 concentrations (124 +/- 14 to 95 +/- 17 mg/dl, p less than 0.05) occurred. Although decreases in low-density lipoprotein (LDL) cholesterol concentrations contributed to the majority of the total decrease observed in plasma cholesterol, IFN did not alter the cholesterol-to-protein ratio of the LDL. When IFN-alpha 2 was discontinued, alterations in plasma lipoproteins returned to pretreatment levels. Plasma triglyceride concentrations were not influenced by IFN treatment nor did administration of IFN decrease very-low-density lipoprotein (VLDL). The overall effect of recombinant (r) IFN-alpha 2 on lipid composition was to reduce LDL and HDL without alteration of VLDL or triglycerides.